NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma.

Overcoming therapeutic resistance in glioblastoma (GBM) is an essential strategy for improving cancer therapy. However, cancer cells possess various evasion mechanisms, such as metabolic reprogramming, which promote cell survival and limit therapy. The diverse metabolic fuel sources that are produced by autophagy provide tumors with metabolic plasticity and are known to induce drug or radioresistance in GBM. This study determined that autophagy, a common representative cell homeostasis mechanism, was upregulated upon treatment of GBM cells with ionizing radiation (IR). Nuclear receptor binding factor 2 (NRBF2)-a positive regulator of the autophagy initiation step-was found to be upregulated in a GBM orthotopic xenograft mouse model. Furthermore, ATP production and the oxygen consumption rate (OCR) increased upon activation of NRBF2-mediated autophagy. It was also discovered that changes in metabolic state were induced by alterations in metabolite levels caused by autophagy, thereby causing radioresistance. In addition, we found that lidoflazine-a vasodilator agent discovered through drug repositioning-significantly suppressed IR-induced migration, invasion, and proliferation by inhibiting NRBF2, resulting in a reduction in autophagic flux in both in vitro models and in vivo orthotopic xenograft mouse models. In summary, we propose that the upregulation of NRBF2 levels reprograms the metabolic state of GBM cells by activating autophagy, thus establishing NRBF2 as a potential therapeutic target for regulating radioresistance of GBM during radiotherapy.

Cardiovascular Profile of Xanthone-Based 1,4 Dihydropyridines Bearing a Lidoflazine Pharmacophore Fragment.

As a follow-up to our previous studies on differently substituted 1,4-dihydropyridines endowed with a peculiar cardiac selectivity, in this paper, a small series of hybrid compounds bearing the pharmacophore fragment of lidoflazine in position 2 or 3 on a 4-(xanthen-9-one)-dihydropyridine core was reported. Lidoflazine was selected due to our promising previously reported data, and the xanthen-9-one substituent was introduced in position 4 of the dihydropyridine scaffold based on the cardiac selectivity observed in several of our studies. The new hybrid compounds were tested to assess cardiac and vascular activities, and the data were evaluated in comparison with those previously obtained for 4-(xanthen-9-one)-dihydropyridines and lidoflazine⁻nifedipine hybrid compounds. The functional studies indicated an interesting peculiar selectivity for the cardiac parameter inotropy, in particular when the lidoflazine fragment was introduced in position 2 of the dihydropyridine scaffold (4a⁻e), and thus a possible preferential binding with the Cav 1.2 isoform of l-type calcium channels, which are mainly involved in cardiac contractility.

Lidoflazine combined with nucleotide precursors increases ATP content and adenosine production in cardiomyocytes.

We have previously identified that the nucleoside transport blocker dipyridamole increases adenosine production but may cause depletion of the nucleotide pool in cardiomyocytes during extended exposure and that this effect was abolished by co-administration of adenine and ribose. The present study aimed to establish whether lidoflazine, a newer generation of nucleoside transport inhibitor with calcium antagonist properties, would cause a similar effect. We conclude that lidoflazine did not affect the nucleotide pool while the combined application of lidoflazine with precursors of nucleotide resynthesis increased ATP concentration and further enhanced adenosine production.

Lidoflazine is a high affinity blocker of the HERG K(+)channel.

Lidoflazine is an antianginal calcium channel blocker that carries a significant risk of QT interval prolongation and ventricular arrhythmia. We investigated whether or not lidoflazine inhibits current through the rapid delayed rectifier K(+) channel alpha subunit (encoded by HERG - human ether-a-go-go-related gene), since this channel has been widely linked to drug-induced QT-prolongation. Lidoflazine inhibited potently HERG current (I(HERG)) recorded from HEK 293 cells stably expressing wild-type HERG (IC(50) of approximately 16 nM). It was approximately 13-fold more potent against HERG than was verapamil under similar conditions. On membrane depolarization, I(HERG) inhibition developed gradually, ruling out closed-channel state dependent inhibition. The effect of command voltage on the drug's action suggested that lidoflazine preferentially inhibits activated/open HERG channels. The S6 mutation Y652A largely eliminated the inhibitory action of lidoflazine, whilst the F656A mutation also reduced blocking potency. We conclude: first, that lidoflazine produces high affinity blockade of the alpha subunit of the HERG channel by binding to aromatic amino acid residues within the channel pore and, second, that this is likely to represent the molecular mechanism of QT interval prolongation by this drug.

Síntese de derivados diidrofurânicos, `alfa'-metilenolactonas e dos princípios ativos lidoflazina e PR-608 / Synthesis of dihydrofurans derivatives, alpha-methylene lactones and biologically actives lidoflazine and PR608

A primeira parte deste trabalho refere-se às reações de ciclofuncionalização, utilizando, como agentes eletrofílicos, indutores de ciclofuncionalização, iodo, brometo de fenilselenenila e tricloreto de p-metoxifeniltelúrio. Na primeira etapa, foram sintetizados compostos 1,3-dicarbonílicos 2,4-dialilsubstituídos, com os grupos alila, crotila e cicloexenila, respectivamente. Os compostos 1,3-dicarbonílicos 2,4-dialilsubstituídos foram submetidos a reações de monociclofuncionalização, produzindo éteres cíclicos funcionalizados, com ligação dupla endocíclica e exocíclica, conforme o esquema ilustrativo a seguir. Com a utilização de iodo em excesso, foi possível obter `alfaï-metilenolactonas ligadas a um anel tetraidrofurânico. A segunda parte deste trabalho refere-se à síntese dos princípios ativos lidoflazina, com atividade vasodilatadora coronariana, e PR-608, com atividade anti-parkinsoniana...

Asphyxiation versus ventricular fibrillation cardiac arrest in dogs. Differences in cerebral resuscitation effects--a preliminary study.

UNLABELLED: We explored the hypothesis that brain damage after cardiac arrest caused by ventricular fibrillation (VF) needs different therapies than that after asphyxiation, which has been studied less thoroughly. In 67 healthy mongrel dogs of both sexes cardiac arrest (at normothermia) by ventricular fibrillation (no blood flow lasting 10 min) or asphyxiation (no blood flow lasting 7 min) was reversed by normothermic external cardiopulmonary resuscitation, followed by intermittent positive-pressure ventilation for 20 h, and intensive care to 96 h. To ameliorate ischemic brain damage, the calcium entry blocker lidoflazine or a solution of free radical scavengers (mannitol and L-methionine in dextran 40) plus magnesium sulphate, was given intravenously immediately upon restoration of spontaneous circulation. Outcome was evaluated as functional deficit, brain creatine kinase (CK) leakage into the cerebrospinal fluid (CSF) and brain morphologic changes. Lidoflazine seemed to improve cerebral outcome after VF but not after asphyxiation. Free radical scavengers plus magnesium sulphate seemed to improve cerebral outcome after asphyxiation, but not after VF. After VF, scattered ischemic neuronal changes in multiple brain regions dominated, and total brain histopathologic damage scores correlated with final neurologic deficit scores at 96 h (r = 0.66) and with peak CK levels in CSF (r = 0.81). After asphyxiation, in addition to the same ischemic neuronal changes, microinfarcts occurred, and there was no correlation between total brain histopathologic damage scores and neurologic deficit scores or CK levels in CSF. CONCLUSIONS: Different mechanisms of cardiac arrest, which cause different morphologic patterns of brain damage, may need different cerebral resuscitation treatments.

Effects of lidoflazine on left ventricular function in patients.

OBJECTIVE: The present study evaluated the effects of the nucleoside transport inhibitor, lidoflazine, at a dose of 1 mg/kg, on left ventricular function. DESIGN: Patients were randomly assigned to receive either lidoflazine or saline in a double-blind manner. SETTING: A university hospital. PARTICIPANTS: The study was performed in 32 patients scheduled for elective coronary artery bypass surgery. INTERVENTIONS: Left ventricular pressures were measured with fluid-filled catheters. Data were digitally recorded during pressure elevation induced by tilt-up of the legs. Transgastric short-axis echocardiographic views of the left ventricle were simultaneously recorded on videotape. Systolic function was evaluated with the slope (Ees, mmHg/mL) of the systolic pressure-volume relationship. Diastolic function was evaluated with the chamber stiffness constant (Kc, mmHg/mL) of the diastolic pressure-volume relationship. Cardiac function was assessed at baseline and after administration of either lidoflazine (group A [n = 16]) or placebo (group B [n = 16]). Data were compared using two-factor analysis of variance. MEASUREMENTS AND MAIN RESULTS: At baseline, diastolic and systolic function were comparable in both groups. Lidoflazine increased Kc from 0.079 +/- 0.015 to 0.125 +/- 0.017 mmHg/mL and decreased Ees from 2.481 +/- 0.213 to 1.217 +/- 0.211 mmHg/mL (p = 0.009 and p = 0.004, respectively). None of these changes occurred when placebo was administered. CONCLUSIONS: Administration of lidoflazine before the start of cardiopulmonary bypass impaired left ventricular systolic function but also increased diastolic stiffness.

1,4-Dihydropyridines bearing a pharmacophoric fragment of lidoflazine.

A series of 1,4-dihydropyridines bearing a pharmacophoric fragment of lidoflazine was synthesized. The compounds were evaluated for inotropic, chronotropic, and calcium antagonist activities. All compounds behave as inotropic and chronotropic agents, except for compounds 4b, 5a, and 5b, which exhibit a rather weak calcium antagonism in vascular smooth muscle (like aorta). Compound 5b is about twofold more potent in decreasing both chronotropy and inotropy, while compound 5c is about fivefold more potent in decreasing inotropy than nifedipine. Moreover, compound 5b is the most potent calcium antagonist derivative of the series.

Neurologic outcomes after cardiac resuscitation.

The purpose of this research utilization article is to familiarize cardiovascular nurses with the Brain Resuscitation Clinical Trials (BRCTs) I and II and discuss the application of these trials to nursing practice. The BRCTs are a series of studies that examine the effects of selected interventions on neurologic outcome after cardiac arrest.

Determinants of early and late results of combined valve operations and coronary artery bypass grafting.

BACKGROUND: Factors determining the outcome of operative correction of valvular abnormalities combined with coronary artery bypass grafting are still incompletely defined. METHODS: Determinants of early and late (more than 90 days) deaths and event-free survival were studied for combined valve operations and coronary artery bypass grafting in 741 patients using multivariate analysis. RESULTS: Ninety-day survival probability was 89% (95% confidence interval, 87% to 92%). Preoperative risk factors for early death were age, female sex, renal failure, New York Heart Association class IV or V, and mitral insufficiency. The operative risk factor was the duration of aortic cross-clamping. Five- and 10-year survival probabilities were 74% (95% confidence interval, 71% to 78%) and 43% (95% confidence interval, 36% to 50%), respectively. Preoperative risk factors for late death were age, preoperative renal failure, New York Heart Association class IV or V, vessel disease, and nonsinus rhythm. Five- and 10-year event-free survival probabilities were 57% (95% confidence interval, 53% to 61%) and 23% (95% confidence interval, 17% to 28%), respectively. Preoperative risk factors for non-event-free survival were age, female sex, reduced left ventricular function, mitral regurgitation, and pacemaker rhythm. CONCLUSION: The demographic factors of age and female sex; the comorbid condition of renal failure; the cardiac conditions of advanced New York Heart Association class, left ventricular function, mitral regurgitation, vessel disease, and cardiac rhythm; and the operative condition of ischemia time are the most important predictors of clinical outcome after combined valve operations and coronary artery bypass grafting.

Old age does not negate good cerebral outcome after cardiopulmonary resuscitation: analyses from the brain resuscitation clinical trials. The Brain Resuscitation Clinical Trial I and II Study Groups.

OBJECTIVE: To assess survival after cardiac arrest and to determine whether age is an independent determinant of late mortality or poor neurologic outcome. DESIGN: Analyses using results of Brain Resuscitation Clinical Trial I (1979 to 1984) and Brain Resuscitation Clinical Trial II (1984 to 1989), two randomized, double-blind studies of outcome following cardiac arrest. SETTING: A multicenter study in 12 acute care hospitals in nine countries (Brain Resuscitation Clinical Trial I), and 24 hospitals in eight countries (Brain Resuscitation Clinical Trial II). PATIENTS: A total of 774 patients who were initially comatose after successful resuscitation from cardiac arrest. The analyses include both in- and out-of-hospital cardiac arrests. RESULTS: The 6-month mortality rate for the entire group was 81%. Mortality rate was 94% for the oldest group (> 80 yrs) compared with 68% for the youngest group (< or = 45 yrs) (p < .01). Other independent predictors of mortality were history of diabetes mellitus, inhospital arrests, arrest time of > 5 mins, history of congestive heart failure, a noncardiac cause of arrest, and cardiopulmonary resuscitation time of > 20 mins. Of the 774 patients, 27% recovered good neurologic function. There was no statistically significant difference in neurologic recovery rates by age. Multivariate analysis showed that independent predictors of good neurologic recovery were: no history of diabetes mellitus, a cardiac cause of arrest, short arrest time, and short cardiopulmonary resuscitation time. CONCLUSION: Increasing age was a factor in postresuscitation mortality, but was not an independent predictor of poor neurologic outcome.

Nucleoside influx and efflux in guinea-pig ventricular myocytes. Inhibition by analogues of lidoflazine.

Adenosine influx and formycin B influx and efflux were characterized in guinea-pig ventricular myocytes at 22 degrees. Transport by both modes was saturable and inhibited by nitrobenzylthioinosine (NBMPR), indicating the presence of an equilibrative NBMPR-sensitive nucleoside transporter in the cardiomyocytes. The kinetic constants for influx and efflux of formycin B, a non-metabolized nucleoside, were similar, suggesting that the nucleoside transporter exhibits symmetrical kinetics (apparent Km 490 +/- 160 and 700 +/- 140 microM; Vmax 6.5 +/- 1.7 and 3.5 +/- 0.3 nmol/10(6) cells per min for influx and efflux, respectively). No evidence was found of either NBMPR-insensitive equilibrative nucleoside transport or sodium-dependent concentrative nucleoside transport. Inhibition of adenosine influx (apparent Km100 +/- 33 microM), by lidoflazine and the analogues mioflazine, soluflazine and R73-335, gave average Ki values of 730, 100, 64 and 2.9 nM, respectively. These compounds also inhibited formycin B efflux with a similar potency to that of adenosine influx. NBMPR-sensitive nucleoside transport was associated with high affinity binding of NBMPR (apparent Kd approximately 1 nM; 9.6 x 10(5) sites/cell). Specific binding of NBMPR was also inhibited by lidoflazine and its analogues. Mioflazine and soluflazine were 20-30-fold more potent at inhibiting NBMPR-sensitive nucleoside influx in guinea-pig erythrocytes than ventricular myocytes, indicating that the potency of some of the compounds studied is tissue dependent.

Central-to-peripheral arterial pressure gradient during cardiopulmonary bypass: relation to pre- and intra-operative data and effects of vasoactive agents.

A significant central-to-peripheral arterial pressure gradient may exist during and after cardiopulmonary bypass (CPB). The etiology and mechanisms of this phenomenon remain controversial. We studied the pressure gradient between aorta, brachial artery and radial artery in 68 patients, scheduled for elective coronary artery bypass surgery. We evaluated whether choice of cardioprotection during CPB (use of cold cardioplegic solution or use of intermittent crossclamping under protection with lidoflazine), and choice of pulsatile or nonpulsatile flow during the course of CPB, affected the magnitude and duration of the systolic pressure gradient. We also studied whether central-to-peripheral pressure gradient was influenced by administration on CPB of different vasoactive drugs with different mode of action: sodium nitroprusside (direct action on the vessels), droperidol (alpha-adrenergic blocking action), ketanserin (5-hydroxytryptamine antagonist) and phenylephrine (selective alpha 1-agonist). It appeared that central-to-peripheral gradient occurred early during CPB and remained constant throughout the course of CPB. The gradient disappeared within 60 min after weaning from CPB. We found the main pressure gradient to occur between the brachial and the radial artery. There was no relation between magnitude of the gradient and sex, weight, length or age of the patient. There was also no relation between magnitude of the pressure gradient and type of cardioprotection, choice of pulsatile vs nonpulsatile flow on CPB and duration of CPB. We also found no relation between pressure gradients and changes in temperature, haematocrit and systemic vascular resistance. The pressure gradient was not affected by any of the vasoactive drugs.

The cardioprotective effects of R 75231 and lidoflazine are not caused by adenosine A1 receptor activation.

The goal of this study was to determine the cardioprotective profile for the nucleoside transport inhibitor 2-(aminocarboxyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis(4- fluorophenyl)pentyl]-1-piperazinylacetamide trihydrochloride-2,5 hydrate (R 75231) in isolated rat hearts and whether its protective effects are caused by adenosine A1 activation. R 75231 increased time to contracture during global ischemia in a concentration-dependent manner (EC25 = 2.6 microM) that was comparable to the structurally related compound lidoflazine (EC25 = 1.2 microM). R 75231 caused only modest improvements in reperfusion contractile function, whereas it profoundly reduced LDH release. The cardioprotective effects of R 75231 were accompanied by preischemic negative inotropy with modest bradycardic effects. Adenosine also increased time to contracture, although it was not very potent (EC25 > 300 microM), and this effect was accompanied by significant preischemic bradycardia without measurable negative inotropic activity. Both the preischemia bradycardia and increase in ischemic time to contracture with adenosine were abolished completely by the A1 blocker 8-cyclopentyl-1,3-dipropylxanthine. The adenosine-induced increase in time to contracture was reversed partially by glybenclamide. Neither the pre- nor postischemic effects of R 75231 were abolished by 8-cyclopentyl-1,3-dipropylxanthine or glybenclamide, except for the preischemic bradycardia. Similar results were observed for lidoflazine. Thus, the cardioprotective effects of R 75231 are not mediated by adenosine A1 receptor activation and, thus, probably are not caused by its activity as a nucleoside transport inhibitor. It may be acting as a calcium antagonist in this model.

K(+)-stimulated 45Ca2+ flux into rat neocortical mini-slices is blocked by omega-Aga-IVA and the dual Na+/Ca2+ channel blockers lidoflazine and flunarizine.

High-threshold neuronal voltage-sensitive Ca2+ channels (VSCCs) have been classified into at least three subtypes, including L, N, and P, based on biophysical and pharmacological criteria. We examined K(+)-induced 45Ca2+ flux into rat neocortical mini-slices to determine which of these subtype(s) might be involved in this phenomenon. Neither the L-type Ca2+ channel antagonist isradipine at 10 microM nor the N-type antagonist omega-conotoxin GVIA at 1 microM were effective antagonists of 45Ca2+ flux in this model. However, the P-type Ca2+ channel antagonist, omega-Aga-IVA, blocked 70% of flux at 200 nM, with an IC50 of 17 nM, strongly implicating P-type Ca2+ channel involvement in K(+)-stimulated Ca2+ entry into mammalian nerve terminals. About 30% of the flux response was resistant to the action of omega-Aga-IVA, suggesting that a still uncharacterized subtype of VSCC is involved in Ca2+ entry into mammalian nerve terminals. Both the omega-Aga-IVA sensitive and insensitive components of 45Ca2+ flux were blocked by the diphenylalkylpiperazines, lidoflazine and flunarizine (IC50 = 6.4 microM and 11 microM, respectively), which have dual Na+/Ca2+ channel blocking actions.

Protective effects of the lazaroid U74500A and lidoflazine on liver preservation with UW solution.

The effect of adding a 21-aminosteroid, U74500A, and a Ca2+ antagonist, lidoflazine, alone and together to UW solution was assessed in a rat liver preservation model. Following preservation, the livers were reperfused using a closed circuit, and the release of hepatocellular enzymes (ASAT, ALAT, and LDH) into the perfusate was determined with increasing time. Both drugs reduced the amount of enzymes lost from the liver. The combination of the two drugs was better than either drug alone. These data suggest that both agents may be of value in organ preservation for clinical liver transplantation.

Simpson's paradox and clinical trials: what you find is not necessarily what you prove.

Expensive clinical trials have become the gold standard for evaluating the efficacy of promising new therapeutic agents. Full exploration of the collected data is routine to maximize the yield of the information available. However, potential methodologic flaws in these extensive analyses may not be appreciated by investigators or readers. One such problem with subgroup analyses is discussed, using hypothetical examples and data from a recently completed clinical trial of brain resuscitation as illustrations.